SRM/MRM Assay for the Ephrin Typa-A Receptor 2 Protein

ABSTRACT

Specific peptides, and derived ionization characteristics of the peptides, from the Ephrin Type-A Receptor 2 (EPHA2) protein are provided that are particularly advantageous for quantifying the EPHA2 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed and are selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue™ reagents and protocol and the EPHA2 protein is quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an EPHA2 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

This application claims the benefit of U.S. Provisional Application No. 61/538,106, filed Oct. 4, 2011, entitled “SRM/MRM Assay for the Ephrin Type-A Receptor 2 Protein,” the contents of which are hereby incorporated by referenced in their entirety.

INTRODUCTION

Specific peptides derived from subsequences of the Ephrin Type-A Receptor 2 protein (referred to herein as EPHA2), are provided. The peptide sequence and fragmentation/transition ions for each peptide are particularly useful in a mass spectrometry-based Selected Reaction Monitoring (SRM) assay, which can also be referred to as a Multiple Reaction Monitoring (MRM) assay. Such assays are referred to herein as SRM/MRM. The use of peptides for quantitative SRM/MRM analysis of the EPHA2 protein is described.

This SRM/MRM assay can be used to measure relative or absolute quantitative levels of one or more of the specific peptides from the EPHA2 protein. This provides a means of measuring the amount of the EPHA2 protein in a given protein preparation obtained from a biological sample by mass spectrometry.

More specifically, the SRM/MRM assay can measure these peptides directly in complex protein lysate samples prepared from cells procured from patient tissue samples, such as formalin fixed cancer patient tissue. Methods of preparing protein samples from formalin-fixed tissue are described in U.S. Pat. No. 7,473,532, the contents of which are hereby incorporated by reference in their entirety. The methods described in U.S. Pat. No. 7,473,532 may conveniently be carried out using Liquid Tissue™ reagents and protocol available from OncoPlexDx (formerly Expression Pathology Inc., Rockville, Md.).

The most widely and advantageously available form of tissues from cancer patients tissue is formalin fixed, paraffin embedded tissue. Formaldehyde/formalin fixation of surgically removed tissue is by far and away the most common method of preserving cancer tissue samples worldwide and is the accepted convention for standard pathology practice. Aqueous solutions of formaldehyde are referred to as formalin. “100%” formalin consists of a saturated solution of formaldehyde (this is about 40% by volume or 37% by mass) in water, with a small amount of stabilizer, usually methanol to limit oxidation and degree of polymerization. The most common way in which tissue is preserved is to soak whole tissue for extended periods of time (8 hours to 48 hours) in aqueous formaldehyde, commonly termed 10% neutral buffered formalin, followed by embedding the fixed whole tissue in paraffin wax for long term storage at room temperature. Thus molecular analytical methods to analyze formalin fixed cancer tissue will be the most accepted and heavily utilized methods for analysis of cancer patient tissue.

Results from the SRM/MRM assay can be used to correlate accurate and precise quantitative levels of the EPHA2 protein within the specific tissue samples (e.g., one or more cancer tissue samples) of the patient or subject from whom the tissue (biological sample) was collected and preserved. This not only provides diagnostic information about the cancer, but also permits a physician or other medical professional to determine appropriate therapy for the patient. For example, such an assay can be designed to diagnose the stage or degree of a cancer and determine a therapeutic agent to which a patient is most likely to respond. Such an assay that provides diagnostically and therapeutically important information about levels of protein expression in a diseased tissue or other patient sample is termed a companion diagnostic assay.

SUMMARY

The assays described herein measure relative or absolute levels of specific unmodified peptides from the EPHA2 protein and also can measure absolute or relative levels of specific modified peptides from the EPHA2 protein. Examples of modifications include phosphorylated amino acid residues (e.g. phosphotyrosine, phosphoserine and phosphothreonine) and glycosylated amino acid residues (e.g. glycosylated asparagine residues) that are present on the peptides.

Relative quantitative levels of the EPHA2 protein are determined by the SRM/MRM methodology for example by comparing SRM/MRM signature peak areas (e.g., signature peak area or integrated fragment ion intensity) of an individual EPHA2 peptide in different samples. Alternatively, it is possible to compare multiple SRM/MRM signature peak areas for multiple EPHA2 signature peptides, where each peptide has its own specific SRM/MRM signature peak, to determine the relative EPHA2 protein content in one biological sample with the EPHA2 protein content in one or more additional or different biological samples. In this way, the amount of a particular peptide, or peptides, from the EPHA2 protein, and therefore the amount of the EPHA2 protein, is determined relative to the same EPHA2 peptide, or peptides, across 2 or more biological samples under the same experimental conditions. In addition, relative quantitation can be determined for a given peptide, or peptides, from the EPHA2 protein within a single sample by comparing the signature peak area for that peptide by SRM/MRM methodology to the signature peak area for another and different peptide, or peptides, from a different protein, or proteins, within the same protein preparation from the biological sample. In this way, the amount of a particular peptide from the EPHA2 protein, and therefore the amount of the EPHA2 protein, is determined relative one to another within the same sample. These approaches generate quantitation of an individual peptide, or peptides, from the EPHA2 protein to the amount of another peptide, or peptides, between samples and within samples wherein the amounts as determined by peak area are relative one to another, regardless of the absolute weight to volume or weight to weight amounts of the EPHA2 peptide in the protein preparation from the biological sample. Relative quantitative data about individual signature peak areas between different samples are normalized to the amount of protein analyzed per sample. Relative quantitation can be performed across many peptides from multiple proteins and the EPHA2 protein simultaneously in a single sample and/or across many samples to gain insight into relative protein amounts, one peptide/protein with respect to other peptides/proteins.

Absolute quantitative levels of the EPHA2 protein are determined by, for example, the SRM/MRM methodology whereby the SRM/MRM signature peak area of an individual peptide from the EPHA2 protein in one biological sample is compared to the SRM/MRM signature peak area of an exogenously added “spiked internal standard.” In one embodiment, the internal standard is a synthetic version of the same exact EPHA2 peptide that contains one or more amino acid residues labeled with one or more heavy isotopes. Suitable isotope labeled internal standards are synthesized so that when analyzed by mass spectrometry each standard generates a predictable and consistent SRM/MRM signature peak that is different and distinct from the native EPHA2 peptide signature peak and which can be used as a comparator peak. Thus when the internal standard is spiked in a known amount into a protein preparation from a biological sample and analyzed by mass spectrometry, the SRM/MRM signature peak area of the native peptide from the sample can be compared to the SRM/MRM signature peak area of the internal standard peptide. This numerical comparison provides either the absolute molarity and/or absolute weight of the native peptide present in the original protein preparation from the biological sample. Absolute quantitative data for fragment peptides are displayed according to the amount of protein analyzed per sample. Absolute quantitation can be performed across many peptides, and thus proteins, simultaneously in a single sample and/or across many samples to gain insight into absolute protein amounts in individual biological samples and in entire cohorts of individual samples.

The SRM/MRM assay method can be used to aid diagnosis of the stage of cancer, for example, directly in patient-derived tissue, such as formalin fixed tissue, and to aid in determining which therapeutic agent would be most advantageous for use in treating that patient. Cancer tissue that is removed from a patient either through surgery, such as for therapeutic removal of partial or entire tumors, or through biopsy procedures conducted to determine the presence or absence of suspected disease, is analyzed to determine whether or not a specific protein, or proteins, and which forms of proteins, are present in that patient tissue. Moreover, the expression level of a protein, or multiple proteins, can be determined and compared to a “normal” or reference level found in healthy tissue. Normal or reference levels of proteins found in healthy tissue may be derived from, for example, the relevant tissues of one or more individuals that do not have cancer. Alternatively, normal or reference levels may be obtained for individuals with cancer by analysis of relevant tissues not affected by the cancer.

Assays of protein levels (e.g., EPHA2 levels) can also be used to diagnose the stage of cancer in a patient or subject diagnosed with cancer by employing the EPHA2 levels. Levels or amounts of proteins or peptides can be defined as the quantity expressed in moles, mass or weight of a protein or peptide determined by the SRM/MRM assay. The level or amount may be normalized to total the level or amount of protein or another component in the lysate analyzed (e.g., expressed in micromoles/microgram of protein or micrograms/microgram of protein). In addition, the level or amount of a protein or peptide may be determined on volume basis, expressed, for example, in micromolar or nanograms/microliter. The level or amount of protein or peptide as determined by the SRM/MRM assay can also be normalized to the number of cells analyzed. Information regarding EPHA2 can thus be used to aid in determining stage or grade of a cancer by correlating the level of the EPHA2 protein (or fragment peptides of the EPHA2 protein) with levels observed in normal tissues.

Once the stage and/or grade, and/or EPHA2 protein expression characteristics of the cancer has been determined, that information can be matched to a list of therapeutic agents (chemical and biological) developed to specifically treat cancer tissue that is characterized by, for example, abnormal expression of the protein or protein(s) (e.g., EPHA2) that were assayed. Matching information from an EPHA2 protein assay to a list of therapeutic agents that specifically targets, for example, the EPHA2 protein or cells/tissue expressing the protein, defines what has been termed a personalized medicine approach to treating disease. The assay methods described herein form the foundation of a personalized medicine approach by using analysis of proteins from the patient's own tissue as a source for diagnostic and treatment decisions.

DETAILED DESCRIPTION

In principle, any predicted peptide derived from EPHA2 protein, prepared for example by digesting with a protease of known specificity (e.g. trypsin), can be used as a surrogate reporter to determine the abundance of EPHA2 protein in a sample using a mass spectrometry-based SRM/MRM assay. Similarly, any predicted peptide sequence containing an amino acid residue at a site that is known to be potentially modified in EPHA2 protein also can be used to assay the extent of modification of EPHA2 protein in a sample.

EPHA2 fragment peptides may be generated by a variety of ways including using the Liquid Tissue™ protocol described, for example, in U.S. Pat. No. 7,473,532. The Liquid Tissue™ protocol and reagents produce peptide samples suitable for mass spectroscopic analysis from formalin fixed paraffin embedded tissue by proteolytic digestion of the proteins in the tissue/biological sample. Suitable reagents and protocols also are commercially available from OncoPlexDx (formerly Expression Pathology Inc., Rockville, Md.).

In the Liquid Tissue™ protocol the tissue/biological sample is heated in a buffer for an extended period of time (e.g., from about 80° C. to about 100° C. for a period of time from about 10 minutes to about 4 hours) to reverse or release protein cross-linking. The buffer employed is a neutral buffer, (e.g., a Tris-based buffer, or a buffer containing a detergent). Following heat treatment the tissue/biological sample is treated with one or more proteases, including but not limited to trypsin, chymotrypsin, pepsin, and endoproteinase Lys-C for a time sufficient to disrupt the tissue and cellular structure of said biological sample and to liquefy the sample. Exemplary conditions for the protease treatment are from about 30 minutes to about 24 hours at a temperature from about 37° C. to about 65° C.). Advantageously, endoproteases, and particularly combinations of two or three endoproteases, used either simultaneously or sequentially, are employed to liquefy the sample. For example, suitable combinations of proteases can include, but are not limited to, combinations of trypsin, endoproteinase Lys-C and chemotrypsin, such as trypsin and endoproteinase Lys-C. The result of the heating and proteolysis is a liquid, soluble, dilutable biomolecule lysate. Advantageously, this liquid lysate is free of solid or particulate matter that can be separated from the lysate by centrifugation.

Surprisingly, it was found that many potential peptide sequences from the EPHA2 protein are unsuitable or ineffective for use in mass spectrometry-based SRM/MRM assays for reasons that are not immediately evident. As it was not possible to predict the most suitable peptides for MRM/SRM assay, it was necessary to experimentally identify modified and unmodified peptides in actual Liquid Tissue™ lysates to develop a reliable and accurate SRM/MRM assay for the EPHA2 protein. While not wishing to be bound by any theory, it is believed that some peptides might, for example, be difficult to detect by mass spectrometry because they do not ionize well, or produce fragments that are not distinct from those generated from other proteins. Peptides may also fail to resolve well in separation (e.g., liquid chromatography), or may adhere to glass or plastic ware, which leads to erroneous results in the EM/MRM assay.

EPHA2 peptides found in various embodiments of this disclosure (e.g., Tables 1 and 2 below) were derived from the EPHA2 protein by protease digestion of all the proteins within a complex Liquid Tissue™ lysate prepared from cells procured from formalin fixed cancer tissue. Unless noted otherwise, in each instance the protease was trypsin. The Liquid Tissue™ lysate was then analyzed by mass spectrometry to determine those peptides derived from the EPHA2 protein that are detected and analyzed by mass spectrometry. Identification of a specific preferred subset of peptides for mass-spectrometric analysis is based on; 1) experimental determination of which peptide or peptides from a protein ionize in mass spectrometry analyses of Liquid Tissue™ lysates, and 2) the ability of the peptide to survive the protocol and experimental conditions used in preparing a Liquid Tissue™ lysate. This latter property extends not only to the amino acid sequence of the peptide but also to the ability of a modified amino acid residue within a peptide to survive in modified form during the sample preparation.

Protein lysates from cells procured directly from formalin (formaldehyde) fixed tissue were prepared using the Liquid Tissue™ reagents and protocol. This entails collecting cells into a sample tube via tissue microdissection followed by heating the cells in the Liquid Tissue™ buffer for an extended period of time. Once the formalin-induced cross linking has been negatively affected, the tissue/cells are then digested to completion in a predictable manner using a protease, such as trypsin. The skilled artisan will recognize that other proteases, and in particular, endoproteases may be used in place of, or in addition to, trypsin. Each protein lysate was used to prepare a collection of peptides by digestion of intact polypeptides with the protease or protease combination. Each Liquid Tissue™ lysate was analyzed (e.g., by ion trap mass spectrometry) to perform multiple global proteomic surveys of the peptides where the data was presented as identification of as many peptides as could be identified by mass spectrometry from all cellular proteins present in each protein lysate. An ion trap mass spectrometer or another form of a mass spectrometer that is capable of performing global profiling for identification of as many peptides as possible from a single complex protein/peptide lysate may be employed. Ion trap mass spectrometers may, however, be the best type of mass spectrometer presently available for conducting global profiling of peptides. Although SRM/MRM assay can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, an advantageous instrument platform for SRM/MRM assay is often considered to be a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single mass spectrometric analysis of a single lysate under the conditions employed, then that list of peptides was collated and used to determine the proteins that were detected in that lysate. That process was repeated for multiple Liquid Tissue™ lysates, and the very large list of peptides was collated into a single dataset. The resulting dataset represents the peptides that can be detected in the type of biological sample that was analyzed (after protease digestion), and specifically in a Liquid Tissue™ lysate of the biological sample, and thus includes the peptides for specific proteins, such as for example the EPHA2 protein.

In one embodiment, the EPHA2 tryptic peptides identified as useful in the determination of absolute or relative amounts of the EPHA2 receptor include either one or both of the peptides of SEQ ID NO:1 and SEQ ID NO:2, the sequences of each of which are shown in Table 1. Each of those peptides was detected by mass spectrometry in Liquid Tissue™ lysates prepared from formalin fixed, paraffin embedded tissue. Thus, either one of the peptides individually or both peptides in combination found in Table 1 are candidates for use in quantitative SRM/MRM assay for the EPHA2 protein in human biological samples, including directly in formalin fixed patient tissue. Table 2 shows additional information regarding the peptides shown in Table 1.

TABLE 1 SEQ ID Peptide sequence SEQ ID NO: 1 TASVSINQTEPPK SEQ ID NO: 2 IDTIAPDEITVSSDFEAR

TABLE 2 Mono Precursor Precursor Transition SEQ ID Peptide sequence Isotopic Charge m/z m/z Ion Type SEQ ID NO: 1 TASVSINQTEPPK 1370.7 2 686.359 813.41 y7 2 686.359 926.494 y8 2 686.359 1013.526 y9 2 690.366 821.424 y7 2 690.366 934.508 y8 2 690.366 1021.54 y9 SEQ ID NO: 2 IDTIAPDEITVSSDFEAR 1977.95 2 989.984 811.358 y7 2 989.984 910.426 y8 2 989.984 1011.474 y9 2 989.984 1124.558 y10 2 989.984 1253.6 y11 2 989.984 1368.627 y12 2 989.984 1465.68 y13

The EPHA2 tryptic peptides listed in Table 1 include those detected from multiple Liquid Tissue™ lysates of multiple different formalin fixed tissues of different human organs including prostate, colon, and breast. Each of those peptides is useful for quantitative SRM/MRM assay of the EPHA2 protein in formalin fixed tissue. Further data analysis of these experiments indicated no preference is observed for any specific peptides from any specific organ site. Thus, each of these peptides is believed to be suitable for conducting SRM/MRM assays of the EPHA2 protein on a Liquid Tissue™ lysate from any formalin fixed tissue originating from any biological sample or from any organ site in the body.

In one embodiment the peptides in Table 1, or both of those peptides are assayed by methods that do not rely upon mass spectroscopy, including, but not limited to, immunological methods (e.g., Western blotting or ELISA). Regardless of how information directed to the amount of the peptide(s) (absolute or relative) is obtained, the information may be employed in any of the methods described herein, including indicating (diagnosing) the presence of cancer in a subject, determining the stage/grade/status of the cancer, providing a prognosis, or determining the therapeutics or treatment regimen for a subject/patient.

Embodiments of the present disclosure include compositions comprising either peptide individually or both of the peptides in Table 1, as further characterized in Table 2. In specific embodiments, the compositions comprise either peptide individually or both peptides in combination where one or both of the peptides are isotopically labeled. Each of the peptides may be labeled with one or more isotopes selected independently from the group consisting of: ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof. Compositions comprising peptides from the EPHA2 protein, whether isotope labeled or not, do not need to contain all of the peptides from that protein (e.g., a complete set of tryptic peptides). In some embodiments the compositions do not contain both peptides in combination from EPHA2, and particularly peptides appearing in Table 1 or Table 2. Compositions comprising peptides may be in the form of dried or lyophized materials, liquid (e.g., aqueous) solutions or suspensions, arrays, or blots.

One consideration for conducting an SRM/MRM assay is the type of instrument that may be employed in the analysis of the peptides. Although SRM/MRM assays can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, the most advantageous instrument platform for SRM/MRM assay is often considered to be a triple quadrupole instrument platform. That type of a mass spectrometer may be considered to be the most suitable instrument for analyzing a single isolated target peptide within a very complex protein lysate that may consist of hundreds of thousands to millions of individual peptides from all the proteins contained within a cell.

In order to most efficiently implement SRM/MRM assay for each peptide derived from the EPHA2 protein it is desirable to utilize information in addition to the peptide sequence in the analysis. That additional information may be used in directing and instructing the mass spectrometer (e.g. a triple quadrupole mass spectrometer) to perform the correct and focused analysis of specific targeted peptide(s) such that the assay may be effectively performed.

The additional information about target peptides in general, and about specific EPHA2 peptides, may include one or more of the mono isotopic mass of the peptide, its precursor charge state, the precursor m/z value, the m/z transition ions, and the ion type of each transition ion. Additional peptide information that may be used to develop an SRM/MRM assay for the EPHA2 protein is shown in Table 2 for the two (2) EPHA2 peptides from the list in Table 1.

The method described below was used to: 1) identify candidate peptides from the EPHA2 protein that can be used for a mass spectrometry-based SRM/MRM assay for the EPHA2 protein, 2) develop individual SRM/MRM assay, or assays, for target peptides from the EPHA2 protein, and 3) apply quantitative assays to cancer diagnosis and/or choice of optimal therapy.

Assay Method 1. Identification of SRM/MRM Candidate Fragment Peptides for the EPHA2 Protein

-   -   a. Prepare a Liquid Tissue™ protein lysate from a formalin fixed         biological sample using a protease or proteases, (that may or         may not include trypsin), to digest proteins     -   b. Analyze all protein fragments in the Liquid Tissue™ lysate on         an ion trap tandem mass spectrometer and identify all fragment         peptides from the EPHA2 protein, where individual fragment         peptides do not contain any peptide modifications such as         phosphorylations or glycosylations     -   c. Analyze all protein fragments in the Liquid Tissue™ lysate on         an ion trap tandem mass spectrometer and identify all fragment         peptides from the EPHA2 protein that carry peptide modifications         such as for example phosphorylated or glycosylated residues     -   d. All peptides generated by a specific digestion method from         the entire, full length EPHA2 protein potentially can be         measured, but preferred peptides used for development of the         SRM/MRM assay are those that are identified by mass spectrometry         directly in a complex Liquid Tissue™ protein lysate prepared         from a formalin fixed biological sample     -   e. Peptides that are specifically modified (phosphorylated,         glycosylated, etc.) in patient tissue and which ionize, and thus         detected, in a mass spectrometer when analyzing a Liquid Tissue™         lysate from a formalin fixed biological sample are identified as         candidate peptides for assaying peptide modifications of the         EPHA2 protein         2. Mass Spectrometry Assay for Fragment Peptides from EPHA2         Protein     -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for         individual fragment peptides identified in a Liquid Tissue™         lysate is applied to peptides from the EPHA2 protein         -   i. Determine optimal retention time for a fragment peptide             for optimal chromatography conditions including but not             limited to gel electrophoresis, liquid chromatography,             capillary electrophoresis, nano-reversed phase liquid             chromatography, high performance liquid chromatography, or             reverse phase high performance liquid chromatography         -   ii. Determine the mono isotopic mass of the peptide, the             precursor charge state for each peptide, the precursor m/z             value for each peptide, the m/z transition ions for each             peptide, and the ion type of each transition ion for each             fragment peptide in order to develop an SRM/MRM assay for             each peptide.         -   iii. SRM/MRM assay can then be conducted using the             information from (i) and (ii) on a triple quadrupole mass             spectrometer where each peptide has a characteristic and             unique SRM/MRM signature peak that precisely defines the             unique SRM/MRM assay as performed on a triple quadrupole             mass spectrometer     -   b. Perform SRM/MRM analysis so that the amount of the fragment         peptide of the EPHA2 protein that is detected, as a function of         the unique SRM/MRM signature peak area from an SRM/MRM mass         spectrometry analysis, can indicate both the relative and         absolute amount of the protein in a particular protein lysate.         -   i. Relative quantitation may be achieved by:             -   1. Determining increased or decreased presence of the                 EPHA2 protein by comparing the SRM/MRM signature peak                 area from a given EPHA2 peptide detected in a Liquid                 Tissue™ lysate from one formalin fixed biological sample                 to the same SRM/MRM signature peak area of the same                 EPHA2 fragment peptide in at least a second, third,                 fourth or more Liquid Tissue™ lysates from least a                 second, third, fourth or more formalin fixed biological                 samples             -   2. Determining increased or decreased presence of the                 EPHA2 protein by comparing the SRM/MRM signature peak                 area from a given EPHA2 peptide detected in a Liquid                 Tissue™ lysate from one formalin fixed biological sample                 to SRM/MRM signature peak areas developed from fragment                 peptides from other proteins, in other samples derived                 from different and separate biological sources, where                 the SRM/MRM signature peak area comparison between the 2                 samples for a peptide fragment are normalized to amount                 of protein analyzed in each sample.             -   3. Determining increased or decreased presence of the                 EPHA2 protein by comparing the SRM/MRM signature peak                 area for a given EPHA2 peptide to the SRM/MRM signature                 peak areas from other fragment peptides derived from                 different proteins within the same Liquid Tissue™ lysate                 from the formalin fixed biological sample in order to                 normalize changing levels of EPHA2 protein to levels of                 other proteins that do not change their levels of                 expression under various cellular conditions.             -   4. These assays can be applied to both unmodified                 fragment peptides and for modified fragment peptides of                 the EPHA2 protein, where the modifications include but                 are not limited to phosphorylation and/or glycosylation,                 and where the relative levels of modified peptides are                 determined in the same manner as determining relative                 amounts of unmodified peptides.         -   ii. Absolute quantitation of a given peptide may be achieved             by comparing the SRM/MRM signature peak area for a given             fragment peptide from the EPHA2 protein in an individual             biological sample to the SRM/MRM signature peak area of an             internal fragment peptide standard spiked into the protein             lysate from the biological sample             -   1. The internal standard is a labeled synthetic version                 of the fragment peptide from the EPHA2 protein that is                 being interrogated. This standard is spiked into a                 sample in known amounts, and the SRM/MRM signature peak                 area can be determined for both the internal fragment                 peptide standard and the native fragment peptide in the                 biological sample separately, followed by comparison of                 both peak areas             -   2. This can be applied to unmodified fragment peptides                 and modified fragment peptides, where the modifications                 include but are not limited to phosphorylation and/or                 glycosylation, and where the absolute levels of modified                 peptides can be determined in the same manner as                 determining absolute levels of unmodified peptides.

3. Apply Fragment Peptide Quantitation to Cancer Diagnosis and Treatment

-   -   a. Perform relative and/or absolute quantitation of fragment         peptide levels of the EPHA2 protein and demonstrate that the         previously-determined association, as well understood in the         field of cancer, of EPHA2 protein expression to the         stage/grade/status of cancer in patient tumor tissue is         confirmed     -   b. Perform relative and/or absolute quantitation of fragment         peptide levels of the EPHA2 protein and demonstrate correlation         with clinical outcomes from different treatment strategies,         wherein this correlation has already been demonstrated in the         field or can be demonstrated in the future through correlation         studies across cohorts of patients and tissue from those         patients. Once either previously established correlations or         correlations derived in the future are confirmed by this assay         then the assay method can be used to determine optimal treatment         strategy

Assessment of EPHA2 protein levels in tissues based on analysis of formalin fixed patient-derived tissue can provide diagnostic, prognostic, and therapeutically-relevant information about each particular patient. In one embodiment, this disclosure describes a method for measuring the level of the EPHA2 protein in a biological sample, comprising detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides in a protein digest prepared from said biological sample using mass spectrometry; and calculating the level of modified or unmodified EPHA2 protein in said sample; and wherein said level is a relative level or an absolute level. In a related embodiment, quantifying one or more EPHA2 fragment peptides comprises determining the amount of the each of the EPHA2 fragment peptides in a biological sample by comparison to a known amount of an added internal standard peptide of known amount, where each of the EPHA2 fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence. In some embodiments the internal standard is an isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof.

The method for measuring the level of the EPHA2 protein in a biological sample described herein (or fragment peptides as surrogates thereof) may be used as a diagnostic indicator of cancer in a patient or subject. In one embodiment, the results from measurements of the level of the EPHA2 protein may be employed to determine the diagnostic stage/grade/status of a cancer by correlating (e.g., comparing) the level of EPHA2 protein found in a tissue with the level of that protein found in normal and/or cancerous or precancerous tissues.

Embodiments

1. A method for measuring the level of the Ephrin Type-A Receptor 2 (EPHA2) protein in a biological sample, comprising detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides in a protein digest prepared from said biological sample using mass spectrometry; and calculating the level of modified or unmodified EPHA2 protein in said sample; and wherein said amount is a relative amount or an absolute amount. 2. The method of embodiment 1, further comprising the step of fractionating said protein digest prior to detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides. 3. The method of embodiment 2, wherein said fractionating step is selected from the group consisting of gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography. 4. The method of any of embodiments 1-3, wherein said protein digest of said biological sample is prepared by the Liquid Tissue™ protocol. 5. The method of any of embodiments 1-3, wherein said protein digest comprises a protease digest. 6. The method of embodiment 5, wherein said protein digest comprises a trypsin digest. 7. The method of any of embodiments 1-6, wherein said mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and/or time of flight mass spectrometry. 8. The method of embodiment 7, wherein the mode of mass spectrometry used is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), and/or multiple Selected Reaction Monitoring (mSRM), or any combination thereof. 9. The method of any of embodiments 1 to 8, wherein the EPHA2 fragment peptide comprises an amino acid sequence as set forth as SEQ ID NO:1 and SEQ ID NO:2. 10. The method of any of embodiments 0-9, wherein the biological sample is a blood sample, a urine sample, a serum sample, an ascites sample, a sputum sample, lymphatic fluid, a saliva sample, a cell, or a solid tissue. 11. The method of embodiment 10, wherein the tissue is formalin fixed tissue. 12. The method of embodiment 10 or 11, wherein the tissue is paraffin embedded tissue. 13. The method of embodiment 10, wherein the tissue is obtained from a tumor. 14. The method of embodiment 13, wherein the tumor is a primary tumor. 15. The method of embodiment 13, wherein the tumor is a secondary tumor. 16. The method of any of embodiments 0 to 15, further comprising quantifying a modified or unmodified EPHA2 fragment peptide. 17. The method of embodiment 16, wherein quantifying the EPHA2 fragment peptide comprises comparing an amount of one or more EPHA2 fragment peptides comprising an amino acid sequence of about 8 to about 45 amino acid residues of EPHA2 as shown in SEQ ID NO:1 or SEQ ID NO:2 in one biological sample to the amount of the same EPHA2 fragment peptide in a different and separate biological sample. 18. The method of embodiment 17, wherein quantifying one or more EPHA2 fragment peptides comprises determining the amount of the each of the EPHA2 fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the EPHA2 fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence. 19. The method of embodiment 18, wherein the internal standard peptide is an isotopically labeled peptide. 20. The method of embodiment 19, wherein the isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof. 21. The method of any of embodiments 1 to 20, wherein detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides in the protein digest indicates the presence of modified or unmodified EPHA2 protein and an association with cancer in the subject. 22. The method of embodiment 21, further comprising correlating the results of said detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides, or the amount of said EPHA2 protein to the diagnostic stage/grade/status of the cancer. 23. The method of embodiment 22, wherein correlating the results of said detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides, or the amount of said EPHA2 protein to the diagnostic stage/grade/status of the cancer is combined with detecting and/or quantifying the amount of other proteins or peptides from other proteins in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer. 24. The method of any one of embodiments 1-23, further comprising selecting for the subject from which said biological sample was obtained a treatment based on the presence, absence, or amount of one or more EPHA2 fragment peptides or the amount of EPHA2 protein. 25. The method of any one of embodiments 1-24, further comprising administering to the patient from which said biological sample was obtained a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent and/or amount of the therapeutic agent administered is based upon amount of one or more modified or unmodified EPHA2 fragment peptides or the amount of EPHA2 protein. 26. The method of embodiments 24 and 25, wherein the treatment or the therapeutic agent is directed to cancer cells expressing the EPHA2 protein. 27. The method of embodiments 1 to 26, wherein the biological sample is formalin fixed tumor tissue that has been processed for quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides employing the Liquid Tissue™ protocol and reagents. 28. The method of any of embodiments 1-27, wherein said one or more modified or unmodified EPHA2 fragment peptides is one or more of the peptides in Table 1. 29. The method of any of embodiments 1-28, comprising quantifying the amount of the peptides in Table 2. 30. A composition comprising either one individually or both in combination of the peptides in Table 1 or antibodies thereto. 

1. A method for measuring the level of the Ephrin Type-A Receptor 2 (EPHA2) protein in a biological sample, comprising detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides in a protein digest prepared from said biological sample using mass spectrometry; and calculating the level of modified or unmodified EPHA2 protein in said sample; and wherein said amount is a relative amount or an absolute amount.
 2. The method of claim 1, further comprising the step of fractionating said protein digest prior to detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides.
 3. The method of claim 2, wherein said fractionating step is selected from the group consisting of gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography.
 4. The method of claim 1, wherein said protein digest of said biological sample is prepared by the Liquid Tissue™ protocol.
 5. The method of claim 1, wherein said protein digest comprises a protease digest.
 6. The method of claim 5, wherein said protein digest comprises a trypsin digest.
 7. The method of claim 1, wherein said mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and/or time of flight mass spectrometry.
 8. The method of claim 7, wherein the mode of mass spectrometry used is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), and/or multiple Selected Reaction Monitoring (mSRM), or any combination thereof.
 9. The method of claim 1, wherein the EPHA2 fragment peptide comprises an amino acid sequence as set forth as SEQ ID NO:1 and SEQ ID NO:2.
 10. The method of claim 1, wherein the biological sample is a blood sample, a urine sample, a serum sample, an ascites sample, a sputum sample, lymphatic fluid, a saliva sample, a cell, or a solid tissue.
 11. The method of claim 10, wherein the tissue is formalin fixed tissue.
 12. The method of claim 10, wherein the tissue is paraffin embedded tissue.
 13. The method of claim 10, wherein the tissue is obtained from a tumor. 14-15. (canceled)
 16. The method of claim 1, further comprising quantifying a modified or unmodified EPHA2 fragment peptide.
 17. The method of claim 16, wherein quantifying the EPHA2 fragment peptide comprises comparing an amount of one or more EPHA2 fragment peptides comprising an amino acid sequence of about 8 to about 45 amino acid residues of EPHA2 as shown in SEQ ID NO:1 or SEQ ID NO:2 in one biological sample to the amount of the same EPHA2 fragment peptide in a different and separate biological sample.
 18. The method of claim 17, wherein quantifying one or more EPHA2 fragment peptides comprises determining the amount of the each of the EPHA2 fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the EPHA2 fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence.
 19. The method of claim 18, wherein the internal standard peptide is an isotopically labeled peptide.
 20. The method of claim 19, wherein the isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof.
 21. The method of claim 1, wherein detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides in the protein digest indicates the presence of modified or unmodified EPHA2 protein and an association with cancer in the subject.
 22. The method of claim 21, further comprising correlating the results of said detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides, or the amount of said EPHA2 protein to the diagnostic stage/grade/status of the cancer.
 23. The method of claim 22, wherein correlating the results of said detecting and/or quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides, or the amount of said EPHA2 protein to the diagnostic stage/grade/status of the cancer is combined with detecting and/or quantifying the amount of other proteins or peptides from other proteins in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer.
 24. The method of claim 1, further comprising selecting for the subject from which said biological sample was obtained a treatment based on the presence, absence, or amount of one or more EPHA2 fragment peptides or the amount of EPHA2 protein.
 25. The method of claim 1, further comprising administering to the patient from which said biological sample was obtained a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent and/or amount of the therapeutic agent administered is based upon amount of one or more modified or unmodified EPHA2 fragment peptides or the amount of EPHA2 protein.
 26. The method of claim 24, wherein the treatment or the therapeutic agent is directed to cancer cells expressing the EPHA2 protein.
 27. The method of claim 1, wherein the biological sample is formalin fixed tumor tissue that has been processed for quantifying the amount of one or more modified or unmodified EPHA2 fragment peptides employing the Liquid Tissue™ protocol and reagents. 28-30. (canceled) 